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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | de Ruijter, Norbert C. A. Roelse, Margriet Jongsma, Maarten A. Vrouwe, Elwin X. |
| Description | Country affiliation: Netherlands Author Affiliation: Roelse M ( Plant Research International, Wageningen UR, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands. margriet.roelse@wur.nl) |
| Abstract | Cell lines expressing recombinant G-protein coupled receptors (GPCRs) are activated by specific ligands resulting in transient [Ca(2+)] rises that return to basal levels in 30-60s. Yellow Cameleon 3.6 (YC3.6) is a genetically encoded calcium indicator which can be co-expressed to monitor these cytosolic [Ca(2+)] changes in real-time using Förster (Fluorescence) resonance energy transfer (FRET). On this basis, we designed the prototype of a generic microfluidic biosensor of GPCR activation, imaging [Ca(2+)] changes in recombinant human HEK293 cells, which express a combination of a GPCR (Neurokinin 1) and YC3.6. An internal reference for non-specifically induced [Ca(2+)] changes were YC3.6 cells without GPCR but expressing a red fluorescent protein (mCherry) for identification. These cell lines were grown as a mixed population in a flow cell with a volume of ~50µl and a flow cell surface of 170mm(2). Cells were activated by brief exposures to specific and non-specific analytes using an injection valve with a flexible sample volume (tested range 5-100µl) at a flow speed of 100µl/min. A flow cell surface of 0.2mm(2) with 50 cells was imaged every 2-4s to obtain signal kinetics. The lower limit of detection was 30pM Substance P (SP, 2pg/50µl), and reproducible responses to repeated injections every 3min were obtained at 1nM SP. This biosensor was designed for ~50 cells for statistical reasons, but at a lower limit of 1 receptor- and 1 reference-cell, specific ligand detection is still feasible. |
| ISSN | 09565663 |
| Volume Number | 47 |
| e-ISSN | 18734235 |
| Journal | Biosensors and Bioelectronics |
| Language | English |
| Publisher | Elsevier |
| Publisher Date | 2013-09-15 |
| Publisher Place | Great Britain (UK) |
| Access Restriction | One Nation One Subscription (ONOS) |
| Subject Keyword | Biosensing Techniques Methods Calcium Metabolism Microfluidic Analytical Techniques Receptors, Opioid Isolation & Purification Chemistry Calcium-binding Proteins Cell Membrane Cytoplasm Fluorescence Resonance Energy Transfer Hek293 Cells Humans Signal Transduction Journal Article Discipline Biotechnology |
| Content Type | Text |
| Resource Type | Article |
| Subject | Nanoscience and Nanotechnology Medicine Biophysics Biomedical Engineering Biotechnology Electrochemistry |
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